Enzymes responsible for clinical resistance to aminoglycoside antibiotics will be isolated from R-factor resistant E. coli, purified to homogeneity, and characterized with respect to their physical and catalytic properties. The primary method of approach will be to apply steady-state kinetic analyses to gentamicin acetyl transferase I, gentamicin adenylyl transferase, and neomycin-kanamicin phosphotransferase I, to determine their kinetic mechanisms and to tabulate quantitative descriptions of the substrate and inhibitory properties of aminoglycoside antibiotics. The overall objective is to facilitate a search for specific inhibitors of these enzymes to be used to counteract clinical resistance of pathogenic organisms to antibiotic therapy, and to make possible a comparative analysis of these unusual enzymes to more common enzymes which catalyze homologous reactions. Parameters governing the production of these enzymes by bacteria will be examined with a view towards understanding the origin, induction, and expression of R-factor DNA, and ultimately the epidemiology of resistant pathogens.